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Nanoparticle in vitro toxicity studies often report contradictory results with one main reason being insufficient material characterization. In particular the characterization of nanoparticles in biological media remains challenging. Our aim was to provide robust protocols for two of the most commonly applied techniques for particle sizing, i.e. dynamic light scattering (DLS) and differential centrifugal sedimentation (DCS) that should be readily applicable also for users not specialized in nanoparticle physico-chemical characterization. A large number of participants (40, although not all participated in all rounds) were recruited for a series of inter-laboratory comparison (ILC) studies covering many different instrument types, commercial and custom-built, as another possible source of variation. ILCs were organized in a consecutive manner starting with dispersions in water employing well-characterized near-spherical silica nanoparticles (nominal 19 nm and 100 nm diameter) and two types of functionalized spherical polystyrene nanoparticles (nominal 50 nm diameter). At first each laboratory used their in-house established procedures. In particular for the 19 nm silica particles, the reproducibility of the methods was unacceptably high (reported results were between 10 nm and 50 nm). When comparing the results of the first ILC round it was observed that the DCS methods performed significantly worse than the DLS methods, thus emphasizing the need for standard operating procedures (SOPs). SOPs have been developed by four expert laboratories but were tested for robustness by a larger number of independent users in a second ILC (11 for DLS and 4 for DCS). In a similar approach another SOP for complex biological fluids, i.e. cell culture medium containing serum was developed, again confirmed via an ILC with 8 participating laboratories. Our study confirms that well-established and fit-for-purpose SOPs are indispensable for obtaining reliable and comparable particle size data. Our results also show that these SOPs must be optimized with respect to the intended measurement system (e.g. particle size technique, type of dispersant) and that they must be sufficiently detailed (e.g. avoiding ambiguity regarding measurand definition, etc.). SOPs may be developed by a small number of expert laboratories but for their widespread applicability they need to be verified by a larger number of laboratories.

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